Depression and excitation induced in mice by two geometric isomers of (+)13,14-didehydro-20-methyl-carboprostacyclin, FCE 22177 and FCE 22176. Comparison with effects on blood pressure and platelet aggregation

Two geometric isomers of a stable derivative of PGI2 (13,14-didehydro-20-methyl-carbo PGI2), FCE 22177 (5E) and FCE 22176 (5Z), have been injected i.v. in mice (5-400 mcg/Kg) to investigate the effects at CNS level (Irwin's test). The synthetic mixture of the two isomers (5E:5Z = 7:3) was also tested. Isomers 5E and 5Z induced opposite effects: 5E (similar to PGI2) was depressive, 5Z induced stimulation. The mixture showed a strong depressive symptomatology. The two isomers and their mixture induced motor incoordination and impaired the performance of mice in the rotarod test (200 mcg/Kg i.v.). Mice were depressed after 5E and mixture; whilst 5Z induced excitation, in accordance with Irwin's test. 5E and 5Z showed analogous effects on mean blood pressure (hypotension) and heart rate (increase) in normotensive rats, but 5E was 7 times more active than 5Z. The two isomers showed analogous inhibitory effect on guinea-pig platelet aggregation in vitro, but 5E was 30 times more active than 5Z. In conclusion, the two geometric isomers have similar effects on blood pressure, heart rate and platelet aggregation, even if differing quantitatively. In contrast, 5E and 5Z have opposite effects at CNS level on motor function and behaviour. The data suggest the presence of two different sites of action for PGI2 in CNS (Ca2+- and adenylate cyclase-linked?). The two geometric isomers, 5E and 5Z, may be useful to study the PGI2 receptor sites in different tissues.     

Effects of antiplatelet agents on platelet aggregation induced by platelet--activating factor (PAF) in human whole blood.

Impedance aggregometry was used to evaluate the potency of anti-platelet agents on Platelet Activating Factor (PAF)--induced platelet aggregation in citrated human whole blood. Drugs were tested for ability to inhibit maximum aggregation to PAF. Dose response curves were obtained and the concentration of drug producing 50% inhibition of maximum aggregation (ED50) determined. ED50's (microM) for specific PAF antagonists WEB 2086, Ro 19-3704, FR-900452, BN 52021, L-652,731, CV 3988, WEB 2118 and 48740 RP are: 0.39, 2.4, 4.7, 19.5, 21.0, 5.32, 161.0, 924.0, respectively. ED50's for non-specific PAF antagonists, diltiazem, propranolol, ketotifen, procaine HCL, and lidocaine HCL are: 38.0, 56.0, 250.0, 513.0 and 768.0, respectively. Ibuprofen was inactive at 2300 microM. Results are consistent with concept that there are specific receptors on platelets mediating PAF-induced aggregation in whole blood. Aggregation is inhibited potently by specific and competitive PAF receptor antagonists. Whole blood aggregometry may be a valid method for predicting in vivo activity of PAF antagonists.     

Effect of perezone, aminoperezone and their corresponding isomers isoperezone and isoaminoperezone upon in vitro platelet aggregation.

The effect on platelet aggregation of perezone, isoperezone, aminoperezone and isoaminoperezone has been determined in human platelets, using denosinediphosphate (ADP), epinephrine and collagen as inducers. Perezone inhibited ADP- epinephrine- and collagen-induced platelet aggregation; isoperezone, aminoperezone and isoaminoperezone did not. The difference in biological responses could be the result of their structures. While isoperezone, aminoperezone and isoaminoperezone have carbonyl groups in the vicinity of other functional groups, this is not the case for perezone, in which one of the carbonyl groups has an adjacent free position.     

Synergism between PGE1-metabolites(13,14-dihydro-prostaglandin E1, 15-keto prostaglandin E1, 15-keto-13,14-dihydro-prostaglandin E1) and nitric oxide (NO) on platelet aggregation.

A synergistic antiplatelet effect between prostaglandins (PG), cAMP-stimulators and nitric oxide (NO), a cGMP-stimulator, has already been described. Data on a synergism between NO and the metabolites of PGE1, however, are lacking so far. We therefore tested the antiplatelet activity of the metabolites of PGE1 alone and their synergism with NO on human platelets of 8 healthy volunteers in vitro. 13,14-DH-PGE1 (ID 50 = 10.8 ng/ml platelet rich plasma (PRP)) was the only PGE1 metabolite inhibiting the ADP-induced platelet aggregation, its efficacy being 76.4% of the parent compound PGE1 (ID 50 = 8.25 ng/ml PRP). NO (ID 50 = 0.52 microM) also inhibited platelet aggregation. The combined addition of 13,14-dihydro-prostaglandin E1 (13,14-DH-PGE1) and NO caused an additive effect. The other PGE1-metabolites tested, 15-keto prostaglandin (15-K-PGE1) (ID 50 = 16.2. micrograms/ml PRP) and 15-keto-13,14-dihydro-prostaglandin(15-K-13,14-DH-PGE1) (ID 50 = 14.8 micrograms/ml PRP), neither had any relevant antiaggregatory capacity themselves nor a synergistic effect with NO. These findings could be of clinical relevance as a NO-synergism may occur not only with therapeutically administered PGE1 but also with its biologically active metabolite 13,14-DH-PGE1.     

Effects of gaseous anesthetics and ultrashort and short-acting barbiturates on human blood platelet free cytosolic calcium: relevance to their effects on platelet aggregation.

The effects of elevated pressures (to 6 atmospheres absolute (ATA)) of nitrous oxide (N2O) and of xenon (Xe), and barbiturates on platelet free cytosolic calcium ([Ca2+]i) and platelet aggregation were studied. N2O inhibited the ADP-induced rise in [Ca2+]i whereas Xe had no effect. Neither affected basal levels. Pentobarbital and methohexital had little effect on basal or stimulated levels in the presence or "absence" of extracellular Ca2+; but both, at concentrations > 10(-4) M, inhibited platelet aggregation induced by adenosine diphosphate. Thiopental increased basal and stimulated [Ca2+]i when extracellular Ca2+ was present, but not when it was absent, and displayed a bimodal effect with low and high doses being more active than intermediate ones. It also potentiated aggregation. Methitural displayed similar, but nonsignificant, effects. These patterns held for all agents whether or not acetylsalicylic acid was present. Pentobarbital and methohexital inhibited phorbol myristate acetate aggregation in low extracellular calcium and no potentiation was seen with thiopental. In the absence of extracellular Ca2+, no potentiation was observed in stimulated platelets. Potentiation of aggregation previously reported for Xe does not involve increased Ca2+ uptake and did not occur in the absence of extracellular Ca2+. A common mechanism of action for these agents cannot be inferred from their effects on platelet aggregation or [Ca2+]i, as their pharmacological profiles differ markedly. It is evident that their inhibitory properties in this cell are not dependent on extracellular Ca2+, whereas the potentiation observed with pentobarbital, and formerly with Xe, is so dependent.     

Suppressing effects of glucan on micronuclei induced by cyclophosphamide in mice.

The effect of pretreatment with carboxymethylglucan (CMG) on the frequency of micronuclei induced by cyclophosphamide administration in mice was evaluated. Two doses of CMG (50 mg/kg body weight) injected either intraperitoneally 24 h or intravenously 1 h prior to two cyclophosphamide administrations (80 mg/kg) significantly decreased the frequency of micronucleated PCE in bone marrow. Of two evaluated derivatives of carboxymethylglucan, the K3 derivative was most efficient. The results show that it is possible to achieve a suppressive effect of soluble carboxymethylglucan prepared from Saccharomyces cerevisiae against cyclophosphamide mutagenicity. The notion may be useful for glucan's effects against pharmacocarcinogenesis. Therapeutic application of glucan with cyclophosphamide therapy may provide a remarkable decrease of the secondary tumour risk. The utilization of these results for human patients needs to be considered.     

Comparison of the effects of human erythrocyte ghosts and intact erythrocytes on platelet interactions with subendothelium in flowing blood

We investigated whether ghosts behaved similarly to intact erythrocytes to maintain regular primary hemostasis under flow conditions. To this end we performed perfusion experiments with whole blood in which erythrocytes were replaced by pink ghosts, and platelet interaction with the subendothelial surface of a damaged vessel was morphometrically evaluated. The same objective was sought by means of studies with a platelet function analyzer (PFA-100(TM) instrument). Perfusions performed with control blood reconstituted with intact erythrocytes gave rise to 0.4+/-0.2% contact but not spread platelets, 10.8+/-3.4% adhering and spread platelets, 16.3+/-4.6% platelets in thrombi, with 27.5+/-7.4% of the surface covered. Even though the average diameter of the ghosts was smaller than that of intact erythrocytes (5.3 microm vs. 7.7 microm), the values obtained in perfusions performed with ghosts were similar to those of the erythrocyte controls. Studies performed with the PFA-100(TM) analyzer were consistent with those observed in perfusion studies. The viscosity of control blood was compared with that of blood reconstituted with ghosts. At shear rates lower than 450 s(-1), the viscosity of the ghost samples was higher than that of the controls, but the difference progressively decreased as shear rate increased up to 750 s(-1) (3.61+/-0.15 and 3.71+/-0.17 cP, respectively). In conclusion, the results of our study showed that ghosts behaved similarly to intact erythrocytes in maintaining a normal platelet interaction with digested subendothelium, under conditions of moderate shear rate and constant hematocrit (40%). The rheological activity of ghosts, bodies that are metabolically less active, was sufficient for them to satisfactorily act as substitutes for intact erythrocytes in our system.     

Differential inhibitory effects of vitamin E and other antioxidants on prostaglandin synthetase, platelet aggregation and lipoxidase.

Prostaglandin biosynthesis from eicosa-8,11,14-trienoic acid in microsomes from the bovine vesicular gland is inhibited by the antioxidants alpha-naphthol. guaiacol, NDGA and propyl gallate. Prostaglandin biosynthesis in this system is not inhibited by the antioxidants BHT, DL-alpha-tocopherol and Trolox C. Arachidonic acid induced platelet aggregation is inhibited by specifically by alpha-naphthol. guaiacol, NDGA and propyl gallate. Both arachidonic acid induced platelet aggregation and ADP induced platelet aggregation are inhibited non-specifically by the antioxidants BHT, DL-alpha-tocopherol and Trolox C. All antioxidants tested in this study inhibit soybean lipoxidase. Thus alpha-naphthol, NDGA and propyl gallate are non-specific inhibitors of both prostaglandin synthetase and soybean lipoxidase while BHT, DL-alpha-tocopherol and Trolox C are specific inhibitors of soybean lipoxidase alone.     

The effect of ADP, calcium and some inhibitors of platelet aggregation on protein phosphokinases from human blood platelets.

A protein phosphokinase (ATP: protein phosphotransferase EC 2.7.1.37) which is stimulated by 3',5'-cyclic adenosine monophosphate (cyclic AMP) has been partially purified from both the cytoplasmic and membrane fractions of human platelets. The kinetics of both enzymes preparations are similar in respect to cyclic AMP, ATP, ADP and AMP. 5-10-minus 7 M cyclic AMP stimulated both preparations by approximately 100%. Both ADP and AMP at a concentration of 5-10-minus 5 M inhibited protein phosphokinase activity of the soluble and membrane preparation by between 50% and 70%. The response of the two enzyme preparations to calcium differed. 10 mM Ca-2+ inhibited soluble protein phosphokinase activity approximately 80% both in the presence and absence of 5-10 minus 7 M cyclic AMP whereas the same concentrations of Ca-2+ inhibited the membrane-bound enzyme by approximately 60% in the presence of 5-10-minus 7 M cyclic AMP and 40% in the absence of cyclic AMP. This observation may be of importance in understanding the mechanism of platelet aggregation.     

两种发酵食品对糖尿病模型小鼠血糖及血脂的影响

目的：观察诺丽果汁和纳豆两种发酵食品对实验性糖尿病小鼠血糖及血脂的影响。方法：ICR雌性小鼠一次性尾静脉注射四氧嘧啶（55 mg/kg）,72 h后将空腹血糖值≥ 12.00 mmol/L、尿糖呈强阳性（+++）者视为糖尿病小鼠模型。将糖尿病模型小鼠随机分为3组（n=10）：模型（DM）组、诺丽果汁（NJ）组和纳豆（NT）组,另取10只正常ICR雌性小鼠作为正常对照（NC）组。NJ组、NT组分别给予诺丽果汁（25.0 ml/kg）、纳豆（0.6 g/kg）灌胃,其他两组小鼠分别给予生理盐水（25.0 ml/kg）灌胃,连续给药30 d,小鼠自由进食、饮水,记录小鼠饮水量及进食量。末次给药后1.5 h,测定小鼠葡萄糖耐量,经股动脉采血测定小鼠糖化血清蛋白（GSP）、血清胰岛素（Ins）和血脂等指标的变化情况。结果：与NC组比较,DM组小鼠饮水量、进食量、GSP及血清总胆固醇（TC）、甘油三酯（TG）、低密度脂蛋白（LDL）等均显著升高（P<0.01）,葡萄糖耐量、Ins及高密度脂蛋白（HDL）均显著降低（P<0.01）;与DM组比较,NJ组与NT组小鼠GSP、TG及LDL均明显降低（P<0.01,P<0.05）,葡萄糖耐量、Ins和HDL明显升高（P<0.05）。结论：诺丽果汁与纳豆具有降低糖尿病模型小鼠血糖、增加糖耐量及改善血脂的作用,提示二者对糖尿病防治可能具有一定的应用价值。     

Decreased platelet aggregation, increased bleeding time and resistance to thromboembolism in P2Y1-deficient mice.

Platelet activation is characterized by shape change, induction of fibrinogen receptor expression and release of granular contents, leading to aggregation and plug formation. While this response is essential for hemostasis, it is also important in the pathogenesis of a broad spectrum of diseases, including myocardial infarction, stroke and unstable angina. Adenosine 5'-diphosphate (ADP) induces platelet aggregation, but the mechanism for this has not been established, and the relative contribution of ADP in hemostasis and the development of arterial thrombosis is poorly understood. We show here that the purinoceptor P2Y1 is required for platelet shape change in response to ADP and is also a principal receptor mediating ADP-induced platelet aggregation. Activation of P2Y1 resulted in increased intracellular calcium but no alteration in cyclic adenosine monophosphate (cAMP) levels. P2Y1-deficient platelets partially aggregated at higher ADP concentrations, and the lack of P2Y1 did not alter the ability of ADP to inhibit cAMP, indicating that platelets express at least one additional ADP receptor. In vivo, the lack of P2Y1 expression increased bleeding time and protected from collagen- and ADP-induced thromboembolism. These findings support the hypothesis that the ATP receptor P2Y1 is a principal receptor mediating both physiologic and pathological ADP-induced processes in platelets.     

Comparison of the effects of inhibitors of cytochrome P-450-mediated reactions on human platelet aggregation and arachidonic acid metabolism

Metyrapone and SKF-525A, together with amphenone B, a structural analogue of metyrapone, which are all inhibitors of cytochrome P-450-mediated reactions, were shown to inhibit the arachidonic acid-induced aggregation of human platelets. Amphenone B, like metyrapone, exhibited a type II (ligand) binding spectrum with rat liver microsomal cytochrome P-450, in contrast to SKF 525A which is a type I (substrate) binding agent. Independently of their type of binding spectra and of their maximum spectral change, however, the affinity of the three compounds for rat liver cytochrome P-450 showed a close proportional correlation with their platelet aggregation inhibitory potency. All three compounds inhibited the formation of [1-14C]thromboxane B2 from [1-14C]arachidonic acid by human platelets aggregated with collagen. The effect of metyrapone on the remaining labelled products suggested that it is a selective thromboxane synthesis inhibitor, while amphenone B exhibited activity reminiscent of cyclo-oxygenase inhibitors. SKF 525A produced complex effects possibly attributable to cyclo-oxygenase inhibition and enhanced lipid peroxidation, since it also enhanced platelet malonaldehyde formation, which the other two compounds inhibited. These data provide further support for a role of cytochrome P-450 in thromboxane synthesis and platelet aggregation.     

In vivo inhibition of megakaryocyte and platelet production by platelet factor 4 in mice.

The in vivo effect of human platelet factor 4 (PF4) on murine megakaryocytopoiesis and thrombopoiesis was studied. Administration of PF4 induced a dose-dependent decrease in the numbers of megakaryocytes and their progenitor cells (CFU-MK), continuing for 1 week after the injection. However, the size of megakaryocytes and their colonies was not changed following PF4 injection. Platelet levels were significantly decreased at days 3-4. The number of CFU-GM was decreased at days 1-2. White blood cells and hemoglobin were unaffected by PF4. These data indicate that PF4 inhibits megakaryocyte and platelet production in vivo by acting on the early stage of megakaryocyte development.     

Circadian variations of blood pressure in patients with different degrees of hypertension. Changes induced by hypotensive treatment

Arterial pressure was continuously recorded for 24--48 h in 3 normotensive subjects and in 60 hypertensive patients. The greatest variations occurred in those with labile, mild or moderate hypertension compared to those with severe hypertension or normal blood pressure. Atenolol (100-200 mg) administered once or twice daily produced a significant reduction of arterial pressure and a smaller response to the cold pressor test, hand grip and step test in patients with established hypertension, but little change in those with labile hypertension. The evening dose was not followed by a decrease in pressure greater than that observed without treatment, but determined a smaller rise on awaking.     

葛根素对冷激诱导的高血压小鼠的影响

目的：检测葛根素灌胃对冷激诱导的高血压小鼠的血压血脂及肾脏结构的影响。方法：小鼠分为正常对照组、冷激对照组、冷激葛根素饲喂组（2,5,10ms／kg bw3组）（n=12）。以寒冷刺激（4±2℃）建立小鼠高血压模型,每天定时灌胃葛根素治疗,对照组给予聚乙烯吡咯烷酮溶液。连续饲喂18d．检测各组小鼠血压、血脂含量,石蜡切片观察肾脏结构。结果：冷激对照组小鼠血压显著高于正常对照组（P〈0．01）,葛根素饲喂组血压显著低于冷激对照组（P〈0．05）。冷激对照组TG含量显著高于正常对照组（P〈0．01）,而冷激饲喂组TG含量明显降低（P〈0．05）;冷激饲喂组TC含量与冷激对照组相比有所降低但无统计学意义;2mg／kg BW葛根素组LDL-C显著低于冷激对照组（P〈0．01）,各组间HDL-C含量比较无统计学意义,但葛根素饲喂组HDL-C／LDL-C值显著高于冷激对照组。冷激对照组肾小管上皮细胞水肿,内腔极度缩小,肾小球明显胀大,肾小囊腔显著变窄,葛根素饲喂组肾小管水肿基本消失,内腔明显增大,肾小球、肾小囊结构趋于正常。结论：葛根素灌服具有降血压、降血脂、改善病变肾脏结构的作用。     

Effect of salvianolic acids from Radix Salviae miltiorrhizae on regional cerebral blood flow and platelet aggregation in rats.

This study was conducted to observe the effect of salvianolic acids (SA) on regional cerebral blood flow (rCBF) in rats and on platelet aggregation in vitro and in vivo. Cerebral ischemia was produced in rats by occluding of the right middle cerebral artery, together with the right common carotid artery. rCBF was monitored by H2 clearance method with a tissue blood-flow meter. Platelet aggregation induced by collagen, ADP, and AA was measured in vitro and in vivo by platelet aggregometer. Doses of SA at 6 and 10 mg/kg body wt. (i.v.) improved rCBF in rats after ischemia, but had no obvious effect on normal rCBF. In vitro, SA inhibited significantly the platelet aggregation induced by collagen, ADP, and AA with IC50 values of 0.197, 2.22 and 3.29 x 10(3) mg/l, respectively. In vivo, doses of SA at 6 and 10 mg/kg body wt. inhibited significantly the platelet aggregation induced by collagen, and SA at 10 mg/kg body wt. inhibited remarkably platelet aggregation induced by ADP. The results suggest that SA could improve rCBF in the ischemic hemisphere and inhibit platelet aggregation in rats.     

Effects of prostaglandins, cAMP, and changes in cytosolic calcium on platelet aggregation induced by a thromboxane A2 mimic (U46619).

The effects of U46619, a thromboxane mimic, on cytosolic Ca2+ concentration and platelet aggregation were determined in human platelets. Cytosolic Ca2+ concentration was determined by Quin-2 fluorescence and platelet aggregation quantitated with an aggregometer. Addition of U46619 (1 x 10(-7) M) to the platelet suspension produced a rapid increase in cytosolic Ca2+ and platelet aggregation. Pretreatment of platelets with EGTA (3 x 10(-3) M), verapamil (5 x 10(-4) M), a calcium entry blocker, or 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (1 x 10(-3) M), an inhibitor of intracellular Ca2+ release, either blunted or markedly delayed the rate, but not the magnitude, of increase in cytosolic Ca2+ and prevented platelet aggregation by U46619. Pretreatment of platelets with prostaglandin I2 (PGI2) (5 x 10(-8) M), PGD2 (5 x 10(-8) M), PGE1 (5 x 10(-8) M), PGF2 alpha (1 x 10(-5) M), dibutyryl cAMP (5 x 10(-3) M), or forskolin (1 x 10(-6) M) prevented both the increase in cytosolic Ca2+ and the associated platelet aggregation induced by U46619. These data suggest that U46619 may induce platelet aggregation through an increase in cytosolic Ca2+, and that both Ca2+ entry and its release from intracellular storage sites probably contribute to the increase in cytosolic Ca2+. Furthermore, the rate of the increase in cytosolic Ca2+ concentration, as well as the magnitude of the increase, appear to be critical for platelet aggregation induced by U46619. Our data are consistent with the hypothesis that PGs inhibit U46619-induced platelet aggregation by preventing the increase in cytosolic Ca2+, and that these effects may be mediated via an increase in cAMP, since they were induced by PGs and cAMP.